Anti-CRISPR-mediated Acylation and Bioreversible Esterification for Precision Genome Editing

项目总结/摘要 CRISPR/CRISPR相关（Cas）系统的应用 RNA引导的蛋白质（例如，Cas9、Cas 12 a）切割DNA。Cas9和Cas 12 a有许多相似之处， 由于其可编程性，简单性和可编程性， 效率，并应用于基础研究和医学。Cas 12 a通常更有效和特异性 比Cas9然而，由于Cas蛋白的浓度远高于DNA靶的浓度，因此， 基因组编辑，延长的Cas 12 a活性也导致脱靶DNA切割，染色体易位， 和遗传毒性。 为Cas 12 a配备“开-关”开关可以通过缩短Cas 12 a的“开-关”窗口来克服这些挑战。 暴露并增加其特异性-DNA切割的中靶与脱靶比率。我们开发了一个 重新利用CRISPR-Cas抑制剂蛋白的新策略（即，抗CRISPR）选择性地酰化或 具有小分子可去除基团的“笼”Cas 12 a。我们建议将小分子和抗- CRISPR蛋白通过快速、生物可逆和体内相容的方式进入人类细胞， 分别抑制Cas 12 a，提高其特异性。在杰出的麻省理工学院专家的帮助下 和布罗德研究所的设施，我将学习如何使用液相色谱-串联质谱 使用LC-MS/MS技术评估我们的新型Cas 12 a酰化策略的选择性，并在哺乳动物细胞培养中， 通过共聚焦显微镜、下一代测序和计算分析， 抗CRISPR递送和Cas 12 a基因组编辑特异性。总的来说，这项研究将介绍第一个 Cas 12 a赖氨酸残基的位点选择性和可逆酰化方法和新的生物可逆的 酯化策略，用于无痕递送抗CRISPR蛋白，这些蛋白一起将增加基因组- 编辑特异性

PROJECT SUMMARY/ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems use RNA-guided proteins (e.g., Cas9, Cas12a) to cleave DNA. Cas9 and Cas12a share many similarities and have been repurposed for genome editing in human cells because of their programmability, simplicity, and efficiency, with applications in basic research and medicine. Cas12a is generally more efficient and specific than Cas9. However, since the concentration of Cas protein is much higher than that of the DNA target in genome editing, prolonged Cas12a activity also leads to off-target DNA cleavage, chromosomal translocations, and genotoxicity. Equipping Cas12a with an “on-off” switch can overcome these challenges by shortening Cas12a’s window of exposure and increasing its specificity—the on-target to off-target ratio of DNA cleavage. We developed a novel strategy that repurposes a CRISPR-Cas inhibitor protein (i.e., an anti-CRISPR) to selectively acylate or “cage” Cas12a with a small-molecule-removable group. We propose to deliver small molecules and anti- CRISPR proteins into human cells via fast, bioreversible, and in vivo-compatible means to activate and inactivate Cas12a, respectively, and increase its specificity. With the help of experts at the outstanding MIT and Broad Institute facilities, I will learn how to use liquid chromatography–tandem mass spectrometry (LC–MS/MS) to assess the selectivity of our novel Cas12a acylation strategy, and mammalian cell culture, high throughput confocal microscopy, next-generation sequencing, and computational analysis to assess anti-CRISPR delivery and Cas12a genome-editing specificity. Overall, this research will introduce the first approach for the site-selective and reversible acylation of Cas12a lysine residues and a new bioreversible esterification strategy for the traceless delivery of anti-CRISPR proteins that together will increase genome- editing specificity.